The present invention relates to compositions and methods for the promotion of nerve regeneration or prevention or inhibition of neuronal degeneration to ameliorate the effects of injury or disease of the nervous system (NS). In particular, the invention relates to compositions comprising poly-Glu,Tyr and/or activated T cells treated wite poly-Glu,Tyr, to protect central nervous system (CNS) cells from glutamate toxicity, to promote nerve regeneration or to prevent or inhibit neuronal degeneration caused by injury or disease of nerves within the CNS or peripheral nervous system (PNS) of a human subject. The compositions of the present invention may be administered alone or may be optionally administered in any desired combination.
CFA: complete Freund""s adjuvant; CNS: central nervous system; MBP: myelin basic protein; NS: nervous system; PBS: phosphate-buffered saline; pEY: Poly-Glu,Tyr; PNS: peripheral nervous system;; Poly-Glu,Tyr: copolymer poly-Glu50Tyr50, a random heterocopolymer of L-glutamic acid and L-tyrosine,; RGC: retinal ganglion cells.
The nervous system comprises the central (CNS) and the peripheral nervous system (PNS). The CNS is composed of the brain spinal cord and visual system; the PNS consists of all of the other neural elements, namely the nerves and ganglia outside of the brain and spinal cord.
Damage to the nervous system may result from a traumatic injury, such as penetrating trauma or blunt trauma, or a disease or disorder, including but not limited to Alzheimer""s disease, Parkinson""s disease, Huntington""s disease, amyotrophic lateral sclerosis (ALS), diabetic neuropathy, senile dementia, stroke and ischemia.
Maintenance of CNS integrity is a complex xe2x80x9cbalancing actxe2x80x9d in which compromises are struck with the immune system. In most tissues, the immune system plays an essential part in protection, repair, and healing. In the CNS, because of its unique immune privilege, immunological reactions are relatively limited. A growing body of evidence indicates that the failure of the mammalian CNS to achieve functional recovery after injury is a reflection of an ineffective dialog between the damaged tissue and the immune system. For example, the restricted communication between the CNS and blood-borne macrophages affects the capacity of axotomized axons to regrow; transplants of activated macrophages can promote CNS regrowth.
Activated T cells have been shown to enter the CNS parenchyma, irrespective of their antigen specificity, but only T cells capable of reacting with a CNS antigen seem to persist there (Hickey et al, 1991). T cells reactive to antigens of CNS white matter, such as myelin basic protein (MBP), can induce the paralytic disease experimental autoimmune encephalomyelitis (EAE) within several days of their inoculation into naive recipient rats (Ben-Nun, 1981a). Anti-MBP T cells may also be involved in the human disease multiple sclerosis (Ota, K. et al, 1990). However, despite their pathogenic potential, anti-MBP T cell clones are present in the immune systems of healthy subjects (Pette et al, 1990). Activated T cells, which normally patrol the intact CNS, transiently accumulate at sites of central nervous system white matter lesions (Hirschberg et al, 1998).
A catastrophic consequence of CNS injury is that the primary damage is often compounded by the gradual secondary loss of adjacent neurons that apparently were undamaged, or only marginally damaged, by the initial injury (McIntosh, 1993). The primary lesion causes changes in extracellular ion concentrations, elevation of amounts of free radicals, release of neurotransmitters, depletion of growth factors, and local inflammation. These changes trigger a cascade of destructive events in the adjacent neurons that initially escaped the primary injury (Lynch et al, 1994). This secondary damage is mediated by activation of voltage-dependent or agonist-gated channels, ion leaks, activation of calcium-dependent enzymes such as proteases, lipases and nucleases, mitochondrial dysfunction and energy depletion, culminating in neuronal cell death. The widespread loss of neurons beyond the loss caused directly by the primary injury has been called xe2x80x9csecondary degeneration.xe2x80x9d
One of the most common mediators which cause self-propagation of the diseases even when the primary risk factor is removed or attenuated is glutamate, an excitatory amino acid capable of displaying dual activity: playing a pivotal role in normal CNS functioning as an essential neuro-transmitter, but becoming toxic when its physiological levels are exceeded. Elevation of glutamate has been reported in many CNS disorders. In its role as an excitotoxic compound, glutamate is one of the most common mediators of toxicity in acute and chronic (including optic nerve degeneration in glaucoma) degenerative disorders (Pitt et al., 2000). Endogenous glutamate has been attributed to the brain damage occurring acutely after status epilepticus, cerebral ischemia or traumatic brain injury. It may also contribute to chronic neurodegeneration in such disorders as amyotrophic lateral sclerosis and Huntington""s chorea.
Intensive research has been devoted to attenuating the cytotoxic effect of glutamate by the use of locally acting drugs, such as N-methyl-D-aspartate (NMDA)-receptor antagonists. Conventional therapy of this type is often unsatisfactory, however, as in neutralizing the toxic effect it is likely to interfere with the physiological functioning. In humans, such compounds have psychotropic and other side effects that make them unsuitable as therapeutic agents. They also have the disadvantage of interfering with the essential physiological functioning of glutamate as a ubiquitous CNS neurotransmitter. Because glutamate activity is essential for normal physiological functioning, yet is potentially devastating after acute injury or in chronic CNS disorders, any attempt to neutralize its harmful effect must do so without eliminating its essential activity at other sites in the body.
Another tragic consequence of CNS injury is that neurons in the mammalian CNS do not undergo spontaneous regeneration following an injury. Thus, a CNS injury causes permanent impairment of motor and sensory functions.
Spinal cord lesions, regardless of the severity of the injury, initially result in a complete functional paralysis known as spinal shock. Some spontaneous recovery from spinal shock may be observed, starting a few days after the injury and tapering off within three to four weeks. The less severe the insult, the better the functional outcome. The extent of recovery is a function of the amount of initially undamaged tissue minus the loss due to secondary degeneration. Recovery from injury would be improved by neuroprotective treatment that could reduce secondary degeneration. For example, alleviation of the effect of glutamate is a frequent target of neuroprotective drug development. Among the drugs which are being developed for this purpose are N-methyl-D-aspartate (NMDA)-receptor or alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-receptor antagonists. These drugs will inevitably have severe side effects as they interfere with the functioning of NMDA and AMPA receptors, which are crucial for normal CNS activity. One of the most intensely studied NMDA-receptor antagonists is MK801, which provides effective neuroprotection but with severe side effects. In animal models of cerebral ischemia and traumatic brain injury, NMDA and AMPA receptor antagonists protect against acute brain damage and delayed behavioral deficits. Such compounds are undergoing testing in humans, but therapeutic efficacy has yet to be established. Other clinical conditions that may respond to drugs acting on glutamatergic transmission include epilepsy, amnesia, anxiety, hyperalgesia and psychosis (Meldrum, 2000).
In the laboratory of the present inventors, it has recently been discovered that activated T cells that recognize an antigen of the NS of the patient confer neuroprotection. Reference is made to U.S. applications Ser. Nos. 09/218,277 and 09/314,161 and PCT Publication WO 99/60021, the entire contents of which is hereby incorporated herein by reference. More specifically, T cells reactive to MBP were shown to be neuroprotective in rat models of partially crushed optic nerve (see also Moalem et al, 1999a) and of spinal cord injury (see also Hauben et al, 2000). Until recently, it had been thought that immune cells do not participate in NS repair. Furthermore, any immune activity in the context of CNS damage was traditionally considered detrimental for recovery. It was quite surprising to discover that NS-specific activated T cells could be used to protect nervous system tissue from secondary degeneration which may follow damage caused by injury or disease of the CNS or PNS. The mechanism of action of such NS-specific T cells has yet to be discovered, but the massive accumulation of exogenously administered T cells at the site of CNS injury suggests that the presence of T cells at the site of injury plays a prominent role in neuroprotection. It appears, however, that the accumulation, though a necessary condition, is not sufficient for the purpose, as T cells specific to the non-self antigen ovalbumin also accumulate at the site, but have no neuroprotective effect (Hirschberg et al, 1998).
In addition to the NS-specific activated T cells, the above-referenced US applications and PCT publication WO 99/60021 disclose that therapy for amelioration of effects of injury or disease of NS can be carried out also with a natural or synthetic NS-specific antigen such as MAG, S-100, xcex2-amyloid, Thy-1, P0, P2, a neurotransmitter receptor, and preferably human MBP, human proteolipid protein (PLP), and human oligodendrocyte glycoprotein (MOG), or with a peptide derived from an NS-specific antigen such as a peptide comprising amino acids 51-70 of MBP or amino acids 35-55 of MOG.
More recently, it has been discovered in the laboratory of the present inventors that a high molecular weight synthetic basic random copolymer consisting of L-Ala, L-Glu, L-Lys and L-Tyr residues with an average molar fraction of 0.141, 0.427, 0.095 and 0.338, designated Copolymer 1 or Cop 1 and being the active ingredient of COPAXONE(copyright) (Teva Pharmaceuticals Ltd., Israel), a medicament for the treatment of multiple sclerosis, is able to prevent or inhibit neuronal degeneration, or to promote nerve regeneration, in the CNS or PNS, as well as to protect CNS cells from glutamate toxicity. Reference is made to copending U.S. applications Ser. Nos. 09/487,793, 09/620,216, and 09/765,644, the entire contents of which is hereby incorporated herein by reference. More specifically, Cop 1-specific activated T cells were shown to accumulate in both injured and non-injured neuronal tissues and to be protective in the injured optic nerve against the destructive effect of secondary degeneration, and immunization with Cop 1 was shown to protect against glutamate toxicity.
Oral administration of autoantigen in order to obtain xe2x80x9coral tolerancexe2x80x9d has been disclosed for the treatment of various autoimmune diseases. For example, EP 359 783 discloses the oral administration of MBP for the treatment of multiple sclerosis. PCT International Publications WO 91/12816, WO 91/08760 and WO 92/06704 all disclose the treatment of other autoimmune diseases using the oral tolerance method with a variety of autoantigens. Treatment of multiple sclerosis by ingestion or inhalation of Copolymer 1, to achieve suppression of the autoimmune T cell response to myelin antigens, has been disclosed in PCT publication WO 98/30227.
The copolymer poly-Glu50Tyr50, formerly often termed polyGT and hereinafter called pEY, is a random heterocopolymer of L-glutamic acid and L-tyrosine, with an average length of 100 amino acids and a capacity to elicit strong immune response in certain mouse strains (Vidovic et al., 1985; Vidovic and Matzinger, 1988)). More than 20 years ago it was shown that several inbred as well as congenic resistant strains of mice, which fail to respond to pYE, were shown to develop specific plaque-forming cell (PFC) responses when stimulated by YE complexed to an immunogenic carrier such as methylated bovine serum albumin (MBSA), and that pre-immunization with pEY has a tolerogenic effect on the response to YE-MBSA in some mouse strains and this tolerance can be transferred to normal, syngeneic recipients by spleen cells or thymocytes of EY-primed animals (Debre et al., 1975). None of these publications relates, or suggests, the use of pYE for neuroprotection.
Citation or identification of any reference in this section or any other part of this application shall not be construed as an admission that such reference is available as prior art to the invention.
It has now been found by the present inventors that pYE and pYE-activated T cells can protect nerve cells from glutamate toxicity and from undergoing secondary degeneration following spinal cord contusion. We examined the spontaneous appearance of T-cells specific to MBP and T-cells specific to EY in rats after spinal cord contusion. In addition, we used active immunization with pEY to attenuate neuronal degeneration induced by glutamate toxicity or by mechanical injury to the spinal cord.
The present invention thus relates to a method for preventing or inhibiting neuronal degeneration, or for promoting nerve regeneration, in the CNS or PNS, or for protecting CNS cells from glutamate toxicity, which comprises administering to an individual in need thereof an effective amount of an agent selected from the group consisting of (a) poly-Glu,Tyr and (b) T cells which have been activated by poly-Glu,Tyr.
The present invention further provides pharmaceutical compositions comprising a therapeutically effective amount of poly-Glu,Tyr-specific activated T cells and methods for using such compositions to promote nerve regeneration or to prevent or inhibit neuronal degeneration in the CNS or PNS, or for protecting CNS cells from glutamate toxicity, in an amount which is effective to ameliorate the effects of an injury or disease of the NS.
As used herein, the term xe2x80x9cneuroprotectionxe2x80x9d refers to the prevention or inhibition of degenerative effects of injury or disease in the NS, including protection from the secondary neurodegenerative effects which persist even when the primary risk factor is removed or attenuated. This includes protection of both white matter and gray matter.
xe2x80x9cActivated T cellxe2x80x9d as used herein includes (i) T cells that have been activated by exposure to poly-Glu,Tyr and (ii) progeny of such activated T cells.
xe2x80x9cPoly-Glu,Tyr-specific activated T cellsxe2x80x9d as used herein refers to activated T cells having specificity for poly-Glu,Tyr.
The poly-Glu,Tyr-specific activated T cells are used to promote nerve regeneration or to prevent or inhibit the secondary degenerative effects which may follow primary NS injury or the effects of neurodegenerative processes caused by a disease or condition as described hereinafter such as, but not limited to, glaucoma, stroke, ischemia, gunshot, and cerebral damage caused by dangerous sports.
The poly-Glu,Tyr-specific activated T cells serve not only to provide neuroprotection against primary and secondary risk factors associated with myelin (white matter) but also against primary and secondary risk factors associated with the neuronal cell bodies themselves (gray matter) in view of the discovered protection against glutamate toxicity. Thus poly-Glu,Tyr-specific activated T cells are expected to be useful for the purpose of the present invention.
Furthermore, as poly-Glu,Tyr protects from glutamate toxicity, it must also have a regulatory activity, such as by creating regulatory cells or regulatory substances. In view of this regulatory activity, the poly-Glu,Tyr vaccination and the poly-Glu,Tyr-specific activated T cells are expected also to protect white matter and gray matter from damage caused by oxidative stress and other sources of damage to neural cells. In addition, because of this regulatory activity, the present invention can also be used to protect neural cells from autoimmune diseases.
The present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of poly-Glu,Tyr and methods of use of such compositions to promote nerve regeneration or to prevent or inhibit neuronal degeneration in the CNS or PNS, in which the amount is effective to activate T cells in vivo or in vitro, wherein the activated T cells inhibit or ameliorate the effects of an injury or disease of the NS.
In the practice of the invention, therapy for amelioration and treatment of effects of injury or disease comprising administration of poly-Glu,Tyr-specific activated T cells may optionally be in combination with poly-Glu,Tyr.
Additionally, oral administration of poly-Glu,Tyr is effective for neuroprotection after priming with poly-Glu, Tyr administered in adjuvant. Thus, oral poly-Glu,Tyr can be used to boost the activity of the T cells, subsequent to primary activation of such poly-Glu,Tyr, preferably in adjuvant, to build up a critical T cell response immediately after injury.
In another embodiment, cell banks can be established to store poly-Glu,Tyr-sensitized T cells for neuroprotective treatment of individuals at a later time, as needed. In this case, autologous T cells may be obtained from an individual. Alternatively, allogeneic or semi-allogeneic T cells may be stored such that a bank of T cells of each of the most common MHC-class II types are present. In case an individual is to be treated for an injury, preferably autologous stored T cells are used, but, if autologous T cells are not available, then cells should be used which share an MHC type II molecule with the patient, and these would be expected to be operable in that individual. The cells are preferably stored in an activated state after exposure to poly-Glu,Tyr. However, the cells may also be stored in a resting state and activated once they are thawed and prepared for use. The cell lines of the bank are preferably cryopreserved. The cell lines are prepared in any way which is well known in the art. Once the cells are thawed, they are preferably cultured prior to injection in order to eliminate non-viable cells. During this culturing, the cells can be activated or reactivated using the poly-Glu,Tyr, antigen as used in the original activation. Alternatively, activation may be achieved by culturing in the presence of a mitogen, such as phytohemagglutinin (PHA) or concanavalin A (preferably the former). This will place the cells into an even higher state of activation. The few days that it takes to culture the cells should not be detrimental to the patient as the treatment in accordance with the present invention may occur any time up to a week or more after the injury in order to still be effective. Alternatively, if time is of the essence, the stored cells may be administered immediately after thawing.